By the time of the merger, the PCUSA had churches in all 50 states, while the heaviest concentration of UPCNA congregations could be found in western Pennsylvania and parts of Ohio. One institutional expression of the union was the consolidation of two nearby seminaries into the new Pittsburgh Theological Seminary.

In 1946, with cooperation of three other denominations, it formed the United Andean Indian Mission, an agency that sent missionaries to Ecuador.

As had been customary for centuries, the UPCUSA originally held to the Westminster Confession of Faith and catechisms. But, in the 1960s, under the increasing awareness of the diversity of Reformed theologies in Europe that had nurtured the neo-orthodox theological movement, the church appointed a commission to examine and revise, if needed, the traditional standards of doctrine required of ministers and ruling elders. The commission proposed adding several more confessions as doctrinal guides (as opposed to a strictly-enforced "system of Scriptural doctrine," as Westminster was historically conceived) in the UPCUSA Book of Confessions; it also developed a new statement of beliefs, the Confession of 1967, which was heavily influenced by the biblical theology movement of the mid-20th century. Despite strong opposition from conservative evangelicals, much of which dovetailed with their hostility toward the denomination's perceived focus on social action that the Confession of 1967 in particular appeared to endorse, nine-tenths of the presbyteries approved the new documents.

Generally speaking, the UPCUSA (especially its leadership) was a strong supporter of progressive causes, such as civil rights and feminism. Prominent leaders and theologians from the period included Eugene Carson Blake, Robert McAfee Brown, Lloyd John Ogilvie, William Sloane Coffin, and David H. C. Read. Among its members was President Dwight Eisenhower. Like most traditional "mainline" Protestant churches in the U.S., the UPCUSA began to decline numerically in the mid-1960s, reversing a 20-year period of growth on the heels of World War II; some of those losses can be attributed to defections of conservatives (sometimes entire congregations) to evangelical Presbyterian (or other) denominations on the one hand, and on the other, by children who chose for various reasons not to follow their parents' footsteps into church membership. The year 1981 witnessed a number of evangelical congregations leave in order to form the Evangelical Presbyterian Church. In response to this action, that year's General Assembly modified the UPCUSA Book of Order to legally restrain a local church from taking its property to another denomination, without explicit release from its presbytery.

Still, all these unfortunate turns did not deter the UPCUSA from going forward with its ecumenical ambitions, the primary of which was reunion with the Presbyterian Church in the United States (often called, inaccurately, the "Southern" church), which split from the main national body in 1861 at the start of the Civil War. Although the UPCUSA sought from its beginning in putting into effect a merger between the two churches, it was not until the 1970s, when a significant number of conservative PCUS congregations left to form the Presbyterian Church in America, that talks gained strong momentum. A decade's work on the part of both churches resulted in the current-day Presbyterian Church (USA), which began in 1983 (the current PC(USA) should not be confused with the earlier PCUSA, which had the words in its formal title spelled out after the word Church).